Sensitive and rapid HPLC-UV method with back-extraction step for the determination of sildenafil in human plasma

In this work we provided a selective, sensitive and rapid HPLC-UV method for quantification of sildenafil in human plasma. We have adopted a simple liquid-liquid extraction procedure followed a back-extraction in 5% perchloric acid solution. Chromatographic separation was achieved on a BDS C-18Column (150mm×4.6mm, 5μm) using a mobile phase consisted 63% water, 37% acetonitrile and 0.1% triethylamine (pH 7.7). The analysis was detected at 230nm. The achieved lower limit of quantification was 2.00ng/ml. The method showed linear calibration curve over the range of 2.00-200ng/ml. Intra- and inter day precision (CV%) were less than 6.80 and 5.19%, respectively. Whilst intra- and inter day accuracy% were ranged between (98.3 and 105%) and (99.4 and 103%), respectively. Tests confirmed the stability of sildenafil in plasma at room temperature for 24h, during three freeze-thaw cycles, after 24h in autosampler at 10°C and after 60 days in plasma at -30°C. The recovery of sildenafil was greater than 78.4%. The described simple UV method achieved very low limit of quantification and by using simple and inexpensive extraction procedure, complete separation was obtained within short run time. Having demonstrated the validity and novelty of our method, thus it is applicable for the clinical and pharmacokinetic studies of sildenafil in human volunteers especially in laboratories in countries where cost of modern techniques and instrumentation is prohibitive.